Ophthalmic Solution of Smart Supramolecular Peptides to Capture Semaphorin 4D against Diabetic Retinopathy

Abstract Diabetic retinopathy (DR) is the leading cause of vision loss in working age population. Intravitreal injection of anti‐VEGF antibody is widely used in clinical practice. However, about 27% of patients show poor response to anti‐VEGF therapy and about 50% of these patients continue to have macular thickening. Frequent intravitreal injections of antibody may increase the chance of endophthalmitis and cause visual loss or even blindness once happened. Therefore, there is a greatly urgent need for novel noninvasive target to treat DR clinically. Here, the formulation of a smart supramolecular peptide (SSP) eye drop for DR treatment that is effective via specifically identifying and capturing soluble semaphorin 4D (sSema4D), a strongly pro‐angiogenesis and exudates factor, is reported. The SSP nanostructures encapsulate sSema4D so that all biological effects mediated by three receptors of sSema4D are inhibited, thereby significantly alleviating pathological retinal angiogenesis and exudates in DR. Moreover, it is found that combination of SSPs eye drop and anti‐VEGF injection shows better therapeutic effect over anti‐VEGF treatment alone. Overall, SSP eye drop provide an alternative and effective method for noninvasive treatment for DR.

Preparation of peptide-based nanomaterials. SSP-1 or SSP-2 monomers were dissolved in artificial tears at a concentration of 4.0 mM and sonicated for 30 min, followed by quick injection into water at a volume ratio of 1:200 for artificial tears and H 2 O to obtain the nanomaterials solution (20 μM).
TEM for the morphology. The SSP-1 or SSP-2 solution with the concentration of 20 μM (10 μL) was placed dropwise onto a copper mesh for 5 min, then most of the liquid was removed through a filter paper. Uranyl acetate solution (10 μL) were employed to stain the samples for 5 min, followed by drying the spare liquid with the filter. Finally, the copper mesh was washed with 10 μL of deionized water, which was blotted after staining and drying at room temperature. All of the samples were observed by TEM (Tecnai G2 20 S-TWIN) at an accelerating voltage of 120 kV. CD spectra. The CD spectra of SSP-1 or SSP-2 (30 μM) were collected at room temperature using a CD spectrometer (JASCO-1500, Tokyo, Japan) with a cell path length of 1 mm. The measurements were implemented between 190 and 230 nm with a resolution of 1.0 nm and a scanning speed of 300 nm/min. For each measurement, three spectra were collected and averaged. CLSM observation. Sema4D protein was purchased from Novoprotein Ltd. (Suzhou, China). Sema4D protein cultured with SSP-1 or SSP-2 nanomaterials (30 μM) for 4 h were imaged using a Zeiss LSM710 confocal laser scanning microscope (Ultra-VIEWVox, PerkinElmer). The parameters of confocal laser scanning microscope was a 40× objective lens.
Patient. Vitreous humor samples of DR patients were obtained immediately before intravitreal injection of anti-VEGF agents. Control vitreous humor samples were collected from nondiabetic patients receiving surgery for idiopathic macular hole. All of the human vitreous humor samples were collected based on informed consent. The vitreous samples were centrifuged at 4°C, and the supernatants were aliquoted into several sterile tubes and stored at -80°C. The procedure was approved by an ethics committee of Tongji Medical College, Huazhong University of Science and Technology, and strictly followed the Declaration of Helsinki guidelines.
Cell culture. The cerebral cortex of a 3-week-old mouse was minced and digested with 0.1% type II collagenase at 37°C for 45 min. The suspension was centrifuged at 1500g for 15 minutes and the pellet was resuspended in 33% Percoll medium. After the suspension was centrifuged at 1000 g at 4°C for 10 minutes, the microvessel fragments suspended in the middle layer of the culture medium were collected.
Endothelial cells were cultured in Medium 131 (Invitrogen, Carlsbad, CA, USA), and pericytes were cultured in Pericyte Medium (ScienCell, USA). bEnd.3 cells were purchased from ATCC and grown in DMEM with 10% fetal bovine serum.
Preparation method of nanoparticles. The nanomaterials FG-12 and FH-12 were added to a 1:1 mixture of propylene glycol and ethanol solution to form a 2 mM mother liquor. The mother solution was diluted in normal saline at 1:100 dilution to  polyacrylamide gel electrophoresis. The gel-separated proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes. After blocked with 5% skim milk, the membranes were incubated with antibodies against Sema4D (R&D system) or β-actin (Abclonal) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2h at room temperature, and visualized by ECL solution (Abclonal) on a BioSpectrum Imaging System (UVP, Upland, CA, USA). The densitometry was quantified using ImageJ.
Would healing assay. The fused endothelial cells were starved in DMEM without FBS for 6 hours, and a sterile 200 μL pipette tip was used to create a cross linear scratch. After washing the floating cells with PBS, the cell culture medium of different stimulation groups was changed to DMEM containing mitomycin-C (10 μg/ml) and 0.5% FBS. At 0h and 12h, pictures of the wounds of endothelial cells were collected using a microscope. Use ImageJ to analyze the wound area.

OIR.
OIR model was performed as previously described. The pups and their mothers were exposed to 75% oxygen at 7-12 days after birth, and then exposed to indoor air for another 5 days. At P17, the mice were anesthetized and sacrificed, and the retinas were harvested.
Isolectin B4. Isolectin B4 staining for retinal blood vessel visualization is as described above. After fixing the eyeball with 4% paraformaldehyde (overnight at 4 degrees), the intact retina was peeled off using microscissors and micro tweezers.
After rupture of the membrane with 2% Triton X-100, the retina was placed in the configured isolectin B4 staining (1:100) overnight at 4 degrees. Acquire multiple overlapping (10-20% overlap) images using a fluorescence microscope and merge them together in Adobe Photoshop to visualize the entire retina. Use ImageJ software to quantify the area of pathological neovascularization relative to the total retinal area.
Evans blue assay. Mice were intraperitoneally injected with Evans Blue dye (200 mg/kg) and circulated for 5 hours. The mice were perfused with PBS, and the eyes were fixed with 4% PFA at 4 degrees Celsius for 12 hours, and the retina was The upper and lower eyelids were massaged to make homodisperse on the eye surface. After 12h FITC-labeled FH or FG were instilled, the mice were sacrificed and the eyeballs were wash in the PBS solution. luminescence signal was detected via the In-Vivo FX PRO system (Bruker).
Statistical analysis. Statistical analysis was conducted using Graphpad Prism 8.0 software. All data were expressed by mean ± SD. Unpaired two-tailed Student's t-test (two groups) or one-way ANOVA (more than 2 groups) was used to analyze the statistical significances of the data. Person's correlation analysis was conducted to study the correlation between two variables. It was considered that p < 0.05 was significant, p > 0.05 was not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.